1. Field of the invention
This invention relates to the isolation and purification of a polypeptide growth factor from milk, to synergistic mixtures and pharmaceutical, cosmetic and food compositions comprising it, and to the uses thereof for the promotion and acceleration of wound healing and tissue repair, the suppression of immune responses, the treatment of cancer and the stimulation of growth of mammals and cell cultures. This polypeptide will be referred to herein as Milk Growth Factor (MGF).
2. Background of the invention and prior art
Growth factors can be defined as polypeptides that stimulate cell growth and proliferation at very low concentrations through specific, high-affinity cell surface receptors. This action produces other intracellular signals which result in nutrient uptake, DNA synthesis and cell division, and which eventually lead to tissue growth. Growth factors have been found in a variety of body tissues and fluids, in both the adult and the embryo, and are now believed to be released by most, if not all, cells in culture (see review by Goustin, A. S. et al. (1986), Cancer Research, 46, 1015.) As such, growth factors do not usually function in an endocrine manner but presumably diffuse over short distances through inter-cellular spaces, or act in an autocrine or paracrine fashion.
Milk is one such body fluid that contains factors which stimulate cell growth in culture. A variety of cell types including epithelial cells, normal and transformed fibroblasts, smooth muscle cells and chondrocytes have been shown to proliferate in blood serum-free, milk-supplemented culture medium (Klagsbrun, M. (1980), J.Cell Biol., 84, 808; Steimer, K. S. and Klagsbrun, M. (1981), J.Cell Biol., 88, 294; Sereni, A. and Baserga, R. (1981), Cell Biol. Int. Rep., 5, 338). Such activity has been found in milk and colostrum (the milk expressed during the first few days post partum) obtained from human (Klagsbrun, M. (1978) Proc. Natl. Acad. Sci. USA, 75, 5057.) and bovine sources (Steimer, K. S. et al. (1981), J.Cell Physiol., 109, 223.) In recent years, a number of growth factors have been isolated from milk and purified to homogeneity in an attempt to characterise their structure and biological function.
One of the earliest and better characterised growth factors is Epidermal Growth Factor (EGF), a polypeptide with a molecular weight of about 6 kd and which has growth and proliferative effects on a variety of cells and tissues both in vitro and in vivo. EGF has also been shown to be the major growth promoting agent in human milk (Carpenter, G. (1980), Science, 210, 198; Petrides, P. E. et al., (1985), FEBS Letters 187, 89), bovine milk (Yagi, H. et al., (1986), Acta.Scand.Paed. 75, 233), and murine milk (Beardmore, J. M. and Richards, R. C., (1983) J.Endocrinol. 96, 287).
Two factors with the name of Mammary Derived Growth Factor (MDGF) have also been isolated from milk. Human MDGF-I is a reducing agent-sensitive polypeptide with a molecular weight of about 62 kd and an isoelectric point (pI) of 4.8 (Bano, M. et al., (1985) J.Biol.Chem. 260, 5745). At picomolar levels it stimulates the growth of mammary cells and enhances their levels of collagen production. MDGF-II is also sensitive to disulphide reducing agents, however, has a lower molecular weight of about 17 kd and a pI of 4.4 (Zwiebel, J. A. et al., (1986), Cancer Research, 46, 933). Using high ionic strength eluting buffers, MDGF-II can be resolved from human EGF on a TSK-3000SW gel filtration column although it still competes with radiolabelled .sup.125 I-EGF for binding to the EGF receptor on A431 human epidermoid carcinoma cell membranes. MDGF-II also stimulates the anchorage independant growth of Normal Rat Kidney (NRK) cells in soft agar, the defining characteristic of a Transforming Growth Factor (TGF), and it seems probable that MDGF-II belongs to the TGF-.alpha. family since molecules of the larger TGF-.beta. type do not bind to the EGF receptor. It has also been reported that human milk and bovine milk contain different sets of growth factors. Shing and Klagsbrun (1984, Endocrinol., 115, 273) have isolated three species of growth factor from human milk, which have been named HMGF-I, HMGF-II and HMGF-III. HMGF-III appears to constitute over 75% of the total growth factor activity of human milk as measured by DNA synthesis. HMGF-III has a molecular weight of about 6 kd, a pI of 4.4-4.7, and is insensitive to treatment with reducing agents. Comparative studies have suggested that this molecule is probably EGF. Using the same separation procedures, these workers also showed that bovine colostrum lacks this molecule although it does have a major growth factor component which has a molecular weight of 30-35 kd and is inactivated by treatment with reducing agents. This so-called Bovine Colostrum Growth Factor (BCGF) is biochemically similar to HMGF-II, another molecule which accounts for approximately 20% of the total growth factor activity of human milk.
A Colony Stimulating Factor (CSF) which stimulates in vitro bone marrow cell proliferation and which causes the differentiation of Colony Forming Granulocytic Macrophage pro-genitor cells (CFU-GM) has also been isolated from human milk (Sinha, S. K. and Yunis, A. A. (1983), Biochem. Biophys. Res.Comm. 114, 797). This factor, which is absent in bovine milk or colostrum, is also insensitive to the action of reducing agents. Gel filtration and isoelectric focussing experiments have indicated that it is biochemically distinct from other CSF's and has a molecular weight of 240-250 kd and a pI of 4.4-4.9.
There are a number of other growth factors or related factors which are to be mentioned in connecton with the present invention which were derived from sources other than milk.
Human platelet, human placenta and bovine kidney derived TGF-.beta. molecules are described in International Patent Application W084/01106 and EP 0128849. Patent No. EP-0169016 and U.S. Pat. No. 4,627,982 reports the partial purification of two proteins from bovine demineralized bone (CIF-A and CIF-B), which are co-factors for inducing cartilage formation. CIF-B (TGF-.beta.2) has been found to inhibit inflammatory cell function in vivo (EP 213 776) and to have an inhibiting effect in vitro on the proliferation of tumour cells (EP 271 211). Both of these so-called Cartilage Inducing Factors are active when combined with EGF in the TGF-.beta. assay. One of their factors (CIF-A) has an N-terminal sequence which is identical over the first thirty amino acids to that of human placenta derived TGF-.beta., but which is significantly different to that of CIF-B [see also review articles by Sporn, M. (1986), Science, 233, 532, and Massague, J., Cell 49, 437-438 (1987)].
CIF-B [Seyedin, S. M. et al., (1987) J.Biol.Chem. 262; 1946; EP 169016] is similar, if not identical to two other recently reported growth factors. Cheifetz, S. et al., (1987) Cell, 48: 409) have described a factor which they have isolated from porcine platelets and which they have designated TGF-.beta.2, since it has a different-N-terminal amino acid sequence to the original porcine TGF-.beta. (now being designated TGF-.beta.1 in the scientific literature) isolated from the same source. More recently Wrann, M. et al., (1987) EMBO J. 6: 1633) have isolated a factor from human glioblastoma cells which is immunosuppressive for T-lymphocytes and is designated G-TsF. CIF-B, TGF-.beta.2 and G-TsF have the same amino acid sequence at the N-terminal up to amino acid 19. CIF-B and TGF-.beta.2 have the same molecular weight of about 26 kd, whereas G-TsF has a molecular weight of 12.5 kd. A human TGF type .beta.2 (hTGF-.beta.2) with a molecular weight of 24 kd, and consisting of two disulfide-linked apparently identical polypeptide chains, isolated from the tamoxifen-supplemented human prostatic adenocarcinoma cell line,, has been described by Marquardt, H. et al., J.Biol.Chem., 262, 12127-12131 (1987) and Ikeda, T. et al., Biochem. 26, 2406-2410 (1987).
A growth inhibitor BSC-1 GI, named polyergin, obtained from BSC-1 African green monkey kidney cells, was identified and purified by R. W. Holley et al., Proc. Natl. Acad. Sci. USA, Vol. 75, pp. 1864-1866 (1978), and R. W. Holley et al., ibid. Vol. 77, pp. 5989-5992 (1980). It was shown by R. F. Tucker et al., Science Vol. 226, 705-707 (1984) to have nearly identical biological activity with TGF-.beta.1 and by H. J. Ristow, ibid. Vol. 83, pp. 5531-5533 (1986) to be a strong inhibitor of thymocytic proliferation. The amino acid sequence of BSC-1 GI, as deduced from the cDNA fron a BSC-1 cell cDNA library, was recently found to be identical to the amino acid sequence of TGF-.beta.2 [S. K. Hanks et al., Proc. Natl. Sci. USA, Vol. 85, pp. 79-82 (1988)].
These growth factors hitherto have not been shown to be present in milk.
Although a number of polypeptide growth factors have already been isolated, characterised and cloned, there have been few studies on the activity of these materials in vivo, mainly because of the relatively small amounts available for such experiments. An important indication area for the potential application of the present growth factor is the enhancement of wound healing. Despite the many preparations available for the treatment of wounds there are still large numbers of patients, particularly the elderly, with wounds (including trauma, burns, decubitus and diabetic ulcers) that either heal slowly or fail to heal at all. Such patients present a significant worthwhile target group for a pharmaceutical preparation which would promote and accelerate the wound healing process. Types of small wounds are those in the mouth, especially of the gum, and also those caused e.g. by a razor blade in the face or other parts of the body surface. Stimulation of the growth of mammals, such as in the treatment of dwarfism, and of cell cultures in vitro are also possibilities for the use of the present growth factor.
Surprisingly the present growth factor contains also suppressing activites on certain types of cells, namely those of the immune system and also of cancer cells.
It is quite obvious that a continuous need for the treatment of these indications exists.